Interpretive Summary: Little is known about the microbial makeup and molecular components responsible for biofilm formation on varying substrates and in differing seasonal and geographical environments. Biofilms play a critical role in microbial fermentation in ruminants. However, an ideal model system has not been developed to aid studies on biofilm formation in the rumen. In this study, we developed novel methods and databases for high-throughput identification of peptides and proteins using a marine biofilm model. We demonstrated repeatable method-specific differences in the number of protein identifications and protein coverage and provided criteria to consider for qualitative or quantitative biofilm meta-proteomics experimental design. Our results will facilitate studies in bacterial biofilms associated with plant fiber and food particles in the rumen and the colon of humans and animals. Our methods and databases in the long term will lead to means to alter the microbiome in order to increase cellulose digestion. The acreage currently allocated for cattle rangeland and/or foodstuff production may be reduced, thereby improving operating margins for cattle producers.

Technical Abstract:
The large-scale identification and quantitation of proteins via nano-liquid chromatography (LC)-tandem mass spectrometry (MS/MS) offers a unique opportunity to gain unprecedented insight into the microbial composition and biomolecular activity of true environmental samples. However, in order to realize this potential for marine biofilms, new methods of protein extraction must be developed as many compounds naturally present in biofilms are known to interfere with common proteomic manipulations and LC-MS/MS techniques. In this study, we used amino acid analyses (AAA) and LC-MS/MS to compare the efficacy of three protein extraction methods [using guanidine hydrochloride (GuHCl), sodium dodecyl sulfate (SDS), and phenol], two separation methods [1D-SDS-PAGE-LC and 2D-LC and two digestion methods [in-solution and in-gel] for the metaproteomic analyses of an environmental marine biofilm. The AAA demonstrated that proteins constitute 1.24% of the biofilm wet weight and that the compared methods varied in their protein extraction efficiencies (0.85 – 15.15%). SubsequentLC-MS/MS analyses revealed that the GuHCl / in-solution digestion / 2D-LC method resulted in the greatest number of proteins identified by one or more peptides whereas the phenol / SDS-PAGE-1D-LC/ in-gel digestion method provided the greatest sequence coverage of identified proteins. As expected, metagenomic sequencing of the same biofilm sample enabled the creation of a searchable database that increased the number of protein identifications by 48.7% (=1 peptide) or 54.7% (=2 peptides) when compared to SwissProt database identifications. Taken together, our results provide methods and evidence-based recommendations to consider for qualitative or quantitative biofilm metaproteome experimental design.